Cell-type specific factors bind to regulatory elements located downstream of the TATA-box element in the mouse myelin basic protein (MBP) gene promoter.
Cell-type specific transcription of the myelin basic protein (MBP) gene in primary oligodendrocytes (OL) is regulated by cis-acting regulatory elements located at both upstream and downstream of the TATA-box region of the MBP promoter. To identify cell-type specific factors that bind to the downstream regulatory elements, we utilised DNase I footprinting analysis and gel retardation assays with nuclear extracts from myelin-forming OL as well as a non-myelin forming cell line, C6 glioma (C6) cells. Several regions of DNA were protected from DNAse I digestion by nuclear extracts of both cell types. However, two regions, from -17 to +17 and from +47 to +58 were protected specifically in OL, while three regions, from + 17 to + 22, from +43 to +49 and from +58 to +64 were protected only with C6 nuclear extracts. Inspection of the protected regions for homology with known transcription factor binding sites revealed that sequences at from +47 to +58 and from +56 to +68 showed extensive homology to the negative regulatory element (NRE1), of the mouse renin gene and to the interferon (IFN) consensus sequence of major histocompatibility complex class I genes (MHC I-ICS), respectively. Gel retardation assays using a MHC I-ICS oligonucleotide and transient transfection assays using MBP-CAT constructs were used to study the effect of IFNs on MBP promoter activity in OL and C6 cells. In OL, IFN-alpha/beta caused little induction of CAT activity, but IFN-gamma resulted in a 2-3.5-fold decrease in CAT activity. In contrast, in C6 cells both IFN-alpha/beta and IFN-gamma induced a 1.5-2.5-fold increase in CAT activity. The cooperative effects of factors binding to NREs and ICS may be responsible for the cell-type specific regulation of MBP gene transcription.
Asipu
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School of Biochemistry and Molecular Biology, University of Leeds, UK.
Blair
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GE
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D016899:Interferon-beta / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D051379:Mice; D004676:Myelin Basic Protein / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016385:TATA Box; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D016898:Interferon-alpha; D004676:Myelin Basic Protein; D016899:Interferon-beta; D007371:Interferon-gamma